INDUCTION OF NASOPHARYNGEAL MUCOSAL IMMUNE RESPONSES IN
THE HORSE
John F. Timoney, MVB, PhD, DSc, Keeneland Professor of Infectious Diseases,
Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099
Introduction
Intranasal vaccination has now emerged as a practical approach to the prevention of
equine respiratory infections in N. America; modified live intranasal vaccines against strangles
and equine influenza have become available since 1998 and owe their existence to evidence that
stimulation of protective mucosal immune responses does not result from parenterally inoculated
vaccines but rather requires local induction. Moreover, mucosal immunization often induces a
combination of systemic and local responses associated with production of a greater variety of
immunoglobulin subisotypes and specificities that closely mimic those induced by natural
infection. Finally, vaccines administered intranasally are likely to produce fewer adverse
reactions than when given parenterally and with a lower probability of generating a nonfunctional isotype or high affinity antibody to non-protective epitopes.
Potential mucosal immune induction sites in the equine nasopharynx
Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal
immunity because of its location at the entrance to the respiratory and alimentary tracts. These
tissues in the horse consist mainly of the nasopharyngeal tonsils located in the dorsal
nasopharyngeal recesses and the palatine and lingual tonsils. Less well defined aggregates of
follicular lymphoid tissue are also distributed throughout the nasopharyngeal mucosa. Since the
nasopharyngeal tonsil is potentially an important immune induction target for intranasally
administered vaccines it has been more closely studied (1,2). In brief, its significant features are
a ciliated epithelium that merges with patches of follicle associated antigen sampling epithelium
with M cells that overlie lymphoid tissue consisting of primary or secondary follicles populated
with immunoglobulin producing lymphocytes and dome and parafollicular areas with CD4 and
CD8 positive lymphocytes (Figure 1). CD4 cells also form densely packed clusters in the
subepithelial lamina propria and interfollicular area and probably constitute a population of naive
and memory T cells which provide help for B cells to differentiate and synthesize
immunoglobulin. Intraepithelial lymphocytes are predominantly CD8 but include some B
lymphocytes that are actively extruded into surface secretions where they release IgA, IgG and
IgM. IgA followed by IgGb are quantitatively the most important subisotypes in nasopharyngeal
secretions of adult horses; IgA is absent from those of the foal in the first months of life (3). The
lingual and palatine tonsils differ from the nasopharyngeal tonsil in having parakeratinized or
stratified squamous non-keratinized epithelia respectively (4). Deep crypts increase the available
epithelial surface as much as sixfold. Studies on human nasopharyngeal and palatine tonsils have
demonstrated direct transepithelial access of antigens from the crypt epithelium to underlying
lymphoid tissue. In laboratory rodents some soluble antigens that enter the entire nasopharyngeal
epithelium induce a systemic response or tolerance. Particulate antigens that enter the
nasopharyngeal tonsil are more likely to elicit a secretory IgA response (1). It should be noted
that the lingual and palatine tonsils are preferred sites of attachment of Streptococcus equi
including the avirulent Pinnacle vaccine strain.
Figure 1. Schematic organization of the equine
nasopharyngeal tonsil. LE = lymphoepithelium; FAE =
follicle associated epithelium; ¤ = T lymphocytes (CD4);
 = T lymphocyte (CD8); ? = B lymphocyte; G =
seromucous glands. Courtesy Dr. Pawan Kumar.
FAE
LE
G
Nasopharyngeal mucosal antibody responses to Streptococcus equi and influenza virus
The mucosa of the equine nasopharynx has a complex secretory immune system that
responds to microbial infection by synthesis of specific antibody. The best studied examples of
these infections are strangles and equine influenza (5,6). Mucosal IgA responses to the
antiphagocytic SeM protein of S. equi begin about 2 weeks after infection, peak about 3 weeks
later and persist for 3 to 6 months. Some local SeM specific IgGa and IgGb are also produced but
levels decline rapidly in 3 to 4 weeks. The epitope specificities of locally produced
immunoglobulins may be different than those of serum immunoglobulins – a not unexpected
finding given the well documented independence of mucosal and systemic antibody responses.
Recovery from strangles is associated with immunity to reinfection that persists in approximately
70% of animals for 4 years or longer. Horses challenged shortly after recovery do not make
anamnestic responses to SeM suggesting that convalescent immunity involves immune exclusion.
Interestingly, mucosal responses to S. zooepidemicus in the tonsils do not include antibody
protective against S. equi challenge although the DNA’s of the 2 organisms are almost identical.
Mucosal IgA responses to influenza virus peak on day 16 after infection and return to
baseline by 60 days (5). Low levels of IgGa and IgGb are also present after primary infection for
about 3 months. Horses recovered from natural infection were resistant to challenge 3 months
later. In contrast, horses vaccinated with a parenterally administered vaccine made only IgG(T)
and IgG(C) responses and were not protected. Nevertheless, these horses were apparently primed
and made strong mucosal IgA responses subsequent to challenge.
Intranasal vaccine delivery
Inductive sites in the nasopharynx are an attractive target for new generation vaccines
against respiratory pathogens because of their accessibility, and the absence of chemical barriers
such as acidity and hydrolytic enzymes which may degrade immunogens administered per os to
intestinal mucosal induction sites. However, an intranasal vaccine must be presented in a manner
that will circumvent mucociliary clearance in the nasopharynx and resist mechanical scouring of
the lingual and palatine tonsillar surfaces that accompanies swallowing. The vaccine must not be
affected by naturally occurring polyreactive IgA and must not induce tolerance. It should induce
a memory B and T lymphocyte response. A number of different intranasal delivery systems have
been evaluated in the horse including microparticle encapsulation, mucoadhesive carriers, cholera
toxin chimeras and avirulent salmonellas.
Microparticle encapsulation: Polylactide microspheres have been shown in laboratory
animals to be effective carriers for intranasal delivery of peptides to the mucosal and systemic
immune compartments (7,8). Microencapsulation involves coating of antigen with a
biodegradable polymer such as poly DL-lactide-co-glycolide so that particles less than 5 m are
formed. Particles of this size are endocytosed by M cells and, after antigen release in associated
lymphoid tissue, elicit mucosal and systemic antibodies. However, ponies immunized with 250
g of an immunogenic peptide (SeMF3) of the SeM protein of S. equi on days 0, 7 and 42 made
no detectable serum antibody. SeM specific mucosal IgA responses were detected in 2 vaccinates
on day 21 and in all 3 on day 49. The absence of a systemic response may have been due to
failure of release by antigen from the mucosal compartment. Lack of potency has been frequently
associated with microencapsulation as a mode of mucosal antigen delivery and so the technology
has not been widely embraced (8). An important theoretical advantage of microparticles is that,
unlike living vectors, they do not elicit immune responses to themselves and so repeated
boostering should be possible.
Mucoadhesive compounds: Bioadhesive polymers such as sodium alginate have been
used successfully for mucosal drug delivery and oral application of killed influenza vaccine (8).
Strong mucosal and systemic antibody responses were obtained in this way. Non-polymeric
(food grade) sucrose acetate isobutyrate (SAIB) is a highly viscous excipient which becomes
much less viscous in appropriate solvents including ethanol in which it can be easily aerosolized.
After deposition on the nasopharyngeal mucosa, the solvent evaporates and the SAIB and
dissolved peptides form a sticky film which degrades very slowly. Thus there is prolonged
exposure of the mucosa to antigen trapped in the film. We have used SAIB to stimulate mucosal
and systemic antibody to SeMF3 (9). Adult Thoroughbred mares were inoculated intranasally
with 200 g SeMF3 in 2 mls 75:25 SAIB-ethanol solution on day 0 and again 28 days later.
Control mares were given 200 SeMF3 alone. Ninety-one percent of mares vaccinated with
SeMF3-SAIB had made SeM specific serum and mucosal IgA by day 42. Control mares made
only short-lived mucosal and no serum responses. SAIB was therefore highly effective in
amplifying mucosal and systemic antibody responses to an intranasally administered immunogen.
Interestingly, serum and mucosal antibodies of the same mares showed different patterns of
recognition consistent with the conclusion that SAIB was effective in delivery of SeMF3 to both
the mucosal and systemic immune compartments.
Choleratoxin-SeMF3 chimeras: Cholera toxin (CT) is a potent mucosal immunogen and
adjuvant. The pentameric B (CTB) subunit binds to GM1 gangliosides on epithelial cells and M
cells thereby enhancing its own uptake by mucosal associated lymphoid tissue. The 5 kDa A2
subunit is linked to the central hole formed by the five B subunits and so is potentially a
convenient means of attaching a foreign peptide sequence to the B pentamer (8). Based on this
concept and using recombinant DNA technology we have constructed a CT-SeMF3 chimera that
contains the entire ctB gene and part (A2) of the A sequence encoding the non-toxic region of the
A subunit. One group of ponies was immunized intranasally on days 0, 14, 42, 70 and 126 with
purified CT-SeMF3 and a second group with CTB-A2 alone on days 0, 21 and 56. All ponies
vaccinated with CT-SeMF3 made strong CTB and SeMF3 specific serum IgGb responses after
the first immunization but did not respond to subsequent immunizations as antibody levels slowly
declined (Figure 2). In contrast, SeMF3 specific mucosal IgA responses varied in time of onset
for each pony and were boostable in most ponies. Mucosal responses were delayed in onset
relative to those detected in serum and did not attain the levels seen following strangles. These
unexpected limitations of the CT-chimera approach in the horse are as yet unexplained and are at
variance with reports of experiments with other constructs in laboratory rodents.
Figure 2. SeMF3 specific serum (A) and nasal wash (B) immunoglobulin subisotype responses
of 5 ponies immunized intranasally with CT SeMF3. The arrows on the abscissa indicate
immunization dates. Courtesy Dr. Abhineet Sheoran.
Avirulent salmonella vectors: Avirulent mutants of salmonellas are attractive mucosal
vaccine delivery systems because they stimulate both the mucosal and systemic immune
compartments by penetrating M cells of the Peyer’s patches and then migrating to the spleen and
liver (8). We have demonstrated that intranasal inoculation of ponies with a cya crp mutant
(MGN 707) of Salmonella typhimurium safely elicits strong specific mucosal and systemic
responses. Based on these results and using recombinant DNA technology we prepared a
construct of MGN 707 containing a plasmid with temperature regulated expression of the SeMF3
peptide. Ponies were inoculated intranasally on days 0, 35, 56 and 161 with 6 x 109 cfu MGN
707 pSeMF3 in which expression of SeMF3 had been induced by raising the culture temperature
prior to administration. These ponies made strong SeM specific serum responses and delayed but
strong mucosal IgG responses. In contrast, ponies immunized with non-induced salmonellas
made no detectable mucosal responses although their serum responses to SeM were similar to
those of ponies vaccinated with preinduced salmonellas. This suggests that the vectored peptide
must be expressed and displayed at the time of administration for induction of antigen specific
antibody on the nasopharyngeal mucosa. Both serum and mucosal responses to salmonella
antigens and SeMF3 were boostable allaying the concern raised by other studies in rodents that
preexisting antibody would result in immune exclusion of salmonellas delivered in the booster
inoculum (10).
References
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